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The 13 C-ketoisocaproate ( 13 C-KICA) breath test (BT) has been recently proposed as a non-invasive test for assessing hepatic mitochondrial function. Results of the 13 C-KICA BT can be expressed as different parameters. However, the best parameter for expressing the 13 C-KICA BT result is uncertain which hinders use of the BT in routine clinical practice. We have investigated the repeatability of different parameters of 13 C-KICA BT. Thirteen healthy adult subjects (5 men and 8 women) underwent a 13 C-KICA BT on two occasions separated by a gap of approximately 30 days. There were no significant differences between the repeated measurements for all the test parameters over 30 days. Furthermore, the Bland Altman statistics showed no fixed or proportional bias for any of the test parameters. The cumulative 13 C-dose enrichment over 60 min had the lowest within-subject variability of 12% compared to all other test parameters. The cumulative 13 C-dose enrichment over 60 min could be a very useful parameter for the 13 C-KICA BT to detect impaired hepatic mitochondrial function in patients with chronic liver diseases.

Bile acids (BAs) are endogenous steroids involved in the transport of lipids in bile, acting also as molecular signaling hormones. Primary BAs synthesized in the liver undergo several metabolic pathways in the intestine by gut microbiota to produce secondary BAs. Together with secondary BAs, other metabolites have been recovered from human faeces, including many oxo-BA analogues produced in the colon through oxidation of BA hydroxy groups. However, the complete oxo-BA characterization in biospecimens (particularly intestinal content and faeces) has not been reported yet, hampering the assessment of their potential physiological role. Herein, we have developed and validated a new RP-HPLC-ESI-MS/MS method in negative ionization mode for the simultaneous analysis of 21 oxo-BAs and their 7 metabolic BAs precursors in human faeces. The elution was performed in gradient mode and 28 compounds, including primary, secondary BAs, and their oxo-derivatives, were separated within 50 min at 40 °C column temperature. The method is accurate (bias% <13%), precise (CV% <10%), with limits of quantification (LOQ <30 ng/mL extract samples ), similar for all the studied compounds. The matrix effect does not significantly affect the analysis accuracy, allowing the use of standard solutions for the quantifications, without matrix-matched protocols. Thanks to the high detectability and the relatively high concentration of oxo-BAs (about μg/g wet faeces ), the method does not require a pre-analytical clean-up step. This method was used to identify and quantify oxo-BAs in human faecal samples from healthy subjects, serving as a proof of concept for application in patients with hepatobiliary disease and bacteria overgrowth., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We evaluated the reproducibility of the 13 C-phenylalanine breath test ( 13 C-PheBT). On three separate days, 21 healthy volunteers (11 F and 10 M) underwent 13 C-PheBT with 100 mg l-[1- 13 C]phenylalanine taken orally. Short-term reproducibility was evaluated with paired examinations taken 3 days apart; paired examinations separated by 23 days (median) served for the medium-term reproducibility assessment. Expiratory air was sampled at 19 points throughout 3 h. Determined limited reproducibility of the 13 C-PheBT must be taken into consideration while interpreting the results of this diagnostic tool. The results of this study imply the following conclusions: (i) From among the three parameters examined, the cumulative 13 C recovery area under the curve (AUC) offers much better reproducibility than the maximum momentary 13 C recovery in the expiratory air (D max ) or the time to reach the maximum momentary 13 C recovery (T max ) (ii) Collection of the breath air samples for 2 h results in a much better reproducibility of AUC, than for 1 h only; (iii) Reproducibility of 13 C-PheBT is affected neither by the duration of the time gap between repeated tests nor by gender; (iv) Comparison with data obtained formerly reveals that reproducibility of the 13 C-PheBT is worse than either that of of the 13 C-methacetin ( 13 C-MBT) or the 13 C-alpha-ketoisocaproic acic ( 13 C-KICA-BT) breath tests. This finding will have to be taken into consideration while interpreting the results of this diagnostic tool.

The determination of the acid-base dissociation constants, and thus the pKa values, of α-keto acids such as pyruvic acid is complex because of the existence of these acids in their hydrated and nonhydrated or oxo state. Equilibria involved in the hydration and dehydration of the α-keto group of pyruvic acid and three other α-keto acids, 3-methyl-2-oxobutanoic acid, 4-methyl-2-oxopentanoic acid, and 2-oxo-2-phenylacetic acid, were investigated by proton and carbon nuclear magnetic resonance spectrometry, at constant ionic strength, 0.15, and 25 °C. Dissociation constants for the oxo (pKa(oxo)) and hydrated (pKa(hyd)) acids of each compound were estimated from the change in the degree of hydration with changes in pH and directly from the changes in chemical shifts of various hydrogen and carbons nuclei with pH. α-Keto acids showed greater hydration in their acidic forms than their carboxylate forms. The degree of hydration was sensitive to steric and electronic/resonance factors. As expected, the oxo forms of the acids were stronger acids compared with their hydrated analogs, and their dissociation constants were also sensitive to steric and electronic factors., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

A simple and sensitive method for the simultaneous determination of five active components, D, L-α-hydroxymethionine calcium (HMACa), α-ketovaline calcium (KVCa), D, L-α-ketoisoleucine calcium (KILCa), α-ketoleucine calcium (KLCa) and α-ketophenylalanine calcium (KPACa) of compound α-ketoacid tablet in human urine by ion-pair reversed-phase high performance liquid chromatography (RP-HPLC) was developed and validated. The separation conditions, such as the concentration of ion-pair reagent, the pH value of the mobile phase and the concentration of the buffer were optimized. All the five analytes were separated well on a C18 column (250 mm x 4.6 mm, 5 µm) with diode array detection at 210 nm and the column temperature of 35 °C. The mobile phases were acetonitrile and 20 mmol/L phosphate buffer (containing 15 mmol/L tetrabutylammonium hydroxide; pH 7) at the flow rate of 1. 0 mL/min with gradient elution. The calibration curves for the five components were linear in the range from 20 to 200 mg/L (r≥ 0. 9990). The limits of detection (LODs, S/N= 3) were 3.0, 5. 0, 3. 6, 5.7 and 2. 5 mg/L, and the limits of quantification (LOQs, S/N= 10) were 9. 6, 16.7, 12.0, 19.0 and 8.3 mg/L for HMACa, KVCa, KILCa, KLCa and KPACa, respectively. The intra-day and inter-day precisions were less than 7%, and the average recoveries were between 86.79% and 112. 00% in the human urine with RSDs lower than 9% (n= 5). The method proved precise, specific and reproducible, and can be used for the determination of the five components in urine.

An analytical method which offers accurate determination and identification of eight α-keto acids (α-ketoglutaric acid, pyruvic acid, 4-hydroxyphenylpyruvic acid, 3-methyl-2-oxobutyric acid, α-keto-γ-methylthiobutyric acid, 4-methyl-2-oxovaleric acid, 3-methyl-2-oxovaleric acid, and phenylpyruvic acid) in pork meat and Iberian ham samples is reported. The method utilises a highly selective and sensitive method of multiple reaction monitoring (MRM) by mass spectrometry. The analytical method is simple (although the chemical derivatisation of the α-keto acids with dansylhydrazine is required), precise (<18% RSD), accurate (90-110%), sensitive (0.01-0.34 mg/kg of defatted and freeze-dried meat depending on the α-keto acid) and linear (R>0.99) over several orders of magnitude (until 0.01-146.1 mg/kg of defatted and freeze-dried meat depending on the α-keto acid). Using this methodology, α-keto acids were found to be present in pork meat to a low extent, and their concentration increased when they were determined in Iberian ham. This is the first report of the presence of α-keto acids in both pork meats and Iberian hams., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Organic acids, including keto acids, are key intermediates of central pathways in cellular metabolism. In this study, a comprehensive and reliable method was developed and optimized for the simultaneous measurement of 17 keto acids in various biological samples. The keto acids were converted to solvent extractable forms by ethoximation followed by tert-butyldimethylsilylation for direct analysis by gas chromatography-mass spectrometry in selected ion monitoring mode. The proposed method was precise (0.05-8.3, % RSD) and accurate (-10.5 to 5.3, % RE) with low limit of detection (0.01-0.5ng/mL) and good linearity (r>0.995) in the range of 0.01-5.0μg/mL. This was suitable for profiling analysis of targeted keto acids in human plasma, urine and rat brain tissue., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Asymmetric dimethylarginine (ADMA) is a metabolite of the amino acid L-arginine. It competitively inhibits the enzymatic production of the cell-signaling substance nitric oxide. Therefore, increased levels of ADMA are associated with a range of cardiovascular and other diseases. ADMA is biologically eliminated by direct renal excretion and hydrolysis by the enzyme DDAH. Recently, a further elimination pathway via the transamination by the enzyme AGXT2 to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) has come into the focus of biological research. In this work, we describe an assay for the AGXT2 activity in mouse liver and kidney tissue. It is based on the transformation of isotope-labeled ADMA-d(6) to DMGV-d(6). The quantification of the DMGV-d(6) produced by this reaction in tissue homogenate samples was accomplished by chromatographic separation on a porous graphitic carbon column and tandem mass spectrometric detection. DMGV-d(6) with the deuterium labels in different molecular positions was used as internal standard. The overall production rates of DMGV-d(6) in mice were 195.37 pmol/min/mg total protein in liver and 85.21 pmol/min/mg total protein in kidney tissue, with coefficients of variation of 6.31% and 11.25%, respectively. This method can be applied as a tool for the characterization of the ADMA elimination by the AGXT2 pathway., (Copyright © 2012 John Wiley & Sons, Ltd.)

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate L-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMGV. D(6)-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2., (Copyright © 2011 Elsevier Inc. All rights reserved.)

An enzymatic assay system of D-amino acids was established using the D-amino acid oxidase of Schizosaccharomyces pombe. In this method, the enzyme converts the D-amino acids to the corresponding α-keto acids, which are then reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) in an organic solvent. The resultant fluorescent compounds are separated and quantified by high-performance liquid chromatography (HPLC). Use of an organic solvent following the α-keto acid modification with DMB prevents the non-enzymatic deamination of L-amino acids, which are generally present at much higher concentrations than D-amino acids in biological samples. With this method, D-Glu, D-Asn, D-Gln, D-Ala, D-Val, D-Leu, D-Phe, and D-Ile can be quantified in the order of micromolar, and other D-amino acids except D-Asp can be assayed within a sensitivity range of 50-100 μM. The established enzymatic method was used to analyze the d-amino acid contents in human urine. The concentration of D-Ser obtained using this enzymatic method (223 μM) was in good agreement with that obtained using the conventional HPLC method (198 μM). The enzymatic method also demonstrated that the human urine contained 5.45 μM of d-Ala and 0.91 μM of D-Asn. Both D-amino acids were difficult to be identified using the conventional method, because the large signals from L-amino acids masked those from d-amino acids. The enzymatic method that we have developed can circumvent this problem., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A molecular modeling strategy to screen the capacity of known enzymes to catalyze the reactions of non-native substrates is presented. The binding of pyruvic acid and non-native ketoacids in the active site of pyruvate ferredoxin oxidoreductase was examined using docking analysis, and our results suggest that enzyme-non-native ketoacid-bound species are feasible. Quantum mechanics/molecular mechanics methods were then used to study the geometry of the covalent intermediate formed from the enzyme and the various ketoacids. Finally, quantum mechanical methods were used to study the decarboxylation reaction of 2-keto acids at the mechanistic level. This hierarchical screening ranked the substrates from those that cannot be accommodated by the enzyme (phenyl pyruvate) to those whose conversion rate would most closely approach that of the native substrate (2-ketobutanoic acid and 2-ketovaleric acid). Most notably, our investigation suggests that novel pathways generated using generalized enzyme actions may be screened using the hierarchical approach employed here.

Procedures for the analysis of free alpha-keto acids in human fluids (i.e. plasma, cerebrospinal fluid, urine, etc.) as well as for studying the dynamic free alpha-keto acid pools in differentiated tissues and organ cells have been the subject of growing clinical interest in the study of metabolic regulatory and pathophysiological phenomena. Due to the high instability and polarity of the alpha-keto acids being examined, the development of a quantitative and reproducible analysis of metabolically relevant intracellular alpha-keto acids still presents a substantial methodological challenge. The aim of small sample size, rapid, non-damaging and "metabolism-neutral" cell isolation, careful sample preparation and stability, as well as reproducible analytics technology is not often achieved. Only few of the methods described can satisfy the rigorous demands for an ultra-sensitive, comprehensive and rapid intracellular alpha-keto acid analysis.

A method based on derivatization to fatty acid methyl esters and GC is proposed for the quantitative analysis of hydroxy acids, keto acids and epoxy acids in fats and oils. Isolation of the analytes by solid-phase extraction is proposed to prevent analytical interferences caused by non-altered fatty acids naturally occurring in oils. In addition, hydrogenation is required before the GC analysis to improve repeatability. The analytical method was applied to thermoxidized samples of high linoleic sunflower oil, high oleic sunflower oil and high palmitic sunflower oil. Results showed total levels of these compounds in the order of mg/g of oil in samples with contents of polar compounds ranging from 6.7 to 25.7%. The compounds analyzed constituted major fractions of the oxidized fatty acids.

Excessive fat deposition is the key feature in obesity, which is empowered by cytokines overproduction and stimulation of cell oxidative stress processes, but little is known about energy availability in the form of ATP and mitochondrial function in the obese subjects. Thus, the aim of this study was to evaluate the possible changes in energy metabolism after a 8-weeks balanced-hypocaloric diet in obese subjects by measuring the ATP-content in leukocytes, by assessing 2-keto[1-13C]isocaproate breath test (KICA-BT) parameters related to mitochondrial function and by analyzing inflammatory and oxidative stress biomarkers. All the recruited obese subjects (n = 19) lost body weight after dieting (-5.55 +/- 2.88%). The hypocaloric treatment induced a decrease in leptin levels and lipid peroxidation markers. Interestingly, the ATP content in blood leukocytes increased (49.9 +/- 32.5 vs 36.2 +/- 27.9 pmol/mg prot.; p < 0.05), while KICA tracer mitochondrial oxidation decreased (30.9 +/- 5.9 vs. 33.1 +/- 4.5 % 13C; p < 0.05) after weight loss. These results show that two minimally invasive methods were able to detect changes in mitochondrial function as induced by a hypocaloric diet, which is of great interest in order to understand oxidative processes associated with weight homeostasis as well as to establish newer anti-obesity therapeutic targets by using mitochondrial function markers in vivo.

Natural organic matter (NOM) in the filtrated surface water from a water treatment plant was isolated and fractionated into six types of fractions. These fractions were ozonated at typical ozone dose and reaction time. The yields of several small molecular ozonation by-products, such as, aldehydes, ketones and ketoacids, were monitored after ozonation of the individual NOM fractions. The results showed that formaldehyde and pyruvic acid had the maximal yields among the aldehydes and ketoacids, respectively. Hydrophobic neutral (HON) produced the most significant yield of the ozonation by-products with the specific yield of formaldehyde up to 70.58% of the specific yields and pyruvic acid being 103.2 microg/mg. The hydrophobic NOM fractions produced higher yields of these by-products than the hydrophilic fractions. For example, HON and hydrophobic acid (HOA) produced much higher yields of the ozonation by-products than other fractions. Aldehydes and ketones from HON and HOA and ketoacids from the two fractions accounted for 55.56% and 60% of the ozonation by-products from all the NOM, respectively. Lower production of these by-products was also observed for the basic fractions compared to the acidic and neutral ones. HON and HOA were more biodegradable after ozonation, because their ozonation by-products contributed much higher amount of DOC to the final DOC after ozonation.

The aim of this work was the simultaneous determination of both ketoacids and dicarbonyl compounds in wine. To detect ketoacid compounds in wine, a method based on the quinoxaline derivatives by the reaction with diaminobenzene, currently employed to detect alpha-dicarbonyl compounds, was developed. The quinoxaline derivatives were detected by RP-HPLC with UV detection, which allows the determination of the major dicarbonyl compounds in wine: glyoxal, methylglyoxal, diacetyl and pentane-2,6-dione, and the quinoxaline/quinoxalinol derivatives of alpha-keto-gamma-(methylthio)butyric acid and beta-phenylpyruvic acid (intermediate ketoacid compounds of methional and phenylacetaldehyde) were simultaneously detected by a fluorescence detector. The identification was performed by comparison with standards and also by using LC-MSMS. The levels found in 15 wines analyzed (white wines, Madeira wines, and Port wines) diverge according to the type and the age of the wine. The ketoacid compounds ranged from 0.2 to 5.7 mg/L for alpha-keto-gamma-(methylthio)butyric acid and 0.1 to 9.6 mg/L for beta-phenylpyruvic acid. The quantities observed for dicarbonyl compounds were similar to those already reported.

The products arising from the ozonolysis of oleic acid (cis-9-octadecenoic acid) in solution have been studied using negative ion mode electrospray ionization ion trap mass spectrometry. Oleic acid is an important component of atmospheric organic aerosol and is a key model species in predicting aerosol physical and chemical characteristics. The four predicted reaction products, 1-nonanal, nonanoic acid, 9-oxononanoic acid, and azelaic acid, were all observed in roughly equal yields. In addition to these products a large number of higher molecular weight compounds were detected with m/z ratios of up to 1000 Daltons. Tandem mass spectrometry of these larger ions revealed thatthey represented a complex mixture of linear alpha-acyloxyalkyl hydroperoxides, secondary ozonides, and cyclic diperoxides, formed by reactions between ozonolysis products and Criegee intermediates. These comprise the first directly elucidated structures of large oligomeric species from oleic acid ozonolysis. The degree of oligomerization and hence molecular weight distribution was observed to increase with reaction time in solution.

Low molecular weight dicarboxylic acids (DCAs), ketoacids, and alpha-dicarbonyls have been determined for the PM2.5 samples in a Hong Kong roadway tunnel, using a water extraction followed by a butyl ester and/or dibutyl acetal derivatization technique. For the most wintertime sampling runs, outlet and inlet concentrations of the quantified compounds were found to be quite similar (ca. 10% differences), leading to the conclusion that direct emissions of the organic compounds are insignificant from vehicles in hot-stabilized operations although vehicular emissions can provide important precursors to them. In contrast, a significant concentration increase of most compounds was observed at the outlet station compared to the inlet station in the summertime runs, which might be explained by the secondary production of aerosols in the tunnel. The organic compounds studied comprised a small fraction (<1%) of aerosol organic carbon (OC). In winter, their abundances relative to that of OC in outlet samples were found to be significantly less than those in inlet samples. On the basis of the summer data, apparent secondary production factors of the compounds were calculated, which indicate that adipic and m-phthalic acids can be favorably formed in the tunnel. However, like other DCAs, direct emissions of adipic, m-phthalic, and p-phthalic acids from automobiles are suggested to be insignificant.

We report secondary organic aerosol (SOA) yields from the ozonolysis of alpha-pinene under both dark and UV-illuminated conditions. Exposure to UV light reduces SOA yield by 20-40%, with a maximum reduction in yield coinciding with a minimum in the amount of terpene consumed (15-30 ppb). The data are consistent with a constant absolute reduction in the yield of approximately 0.03. Gas chromatography mass spectrometry analysis of filter samples indicates that the major products found in alpha-pinene SOA include organic acids (e.g., pinic acid), keto acids (e.g., pinonic acid), and hydroxy keto acids (e.g., 10-hydroxypinonic acid). Analysis of filter-based results suggests that yield reduction is a result of the formation of a more volatile product distribution when experiments are conducted in the presence of UV light. These results implythat previous "dark bag" experiments may overestimate SOA generation from monoterpenes and also that SOA generation in the atmosphere may depend significantly on actinic flux.

The effects of combined ozonation and membrane filtration on the removal of the natural organic matter (NOM) and the formation of disinfection by-products (DBPs) were investigated. Ozonation/filtration resulted in a reduction of up to 50% in the dissolved organic carbon (DOC) concentration. Furthermore, humic substances were converted to non-humic substances, with changes in the humic and non-humic substance concentrations of up to -50% and +20%, respectively. Ozonation/filtration resulted in the formation of partially oxidized compounds from NOM that were less reactive with chlorine, decreasing the concentration of simulated distribution system total trihalomethanes (SDS TTHMs) and simulated distribution system halo acetic acids (SDS HAAs) by up to 80% and 65%, respectively. Reducing the molecular weight cut-off (MWCO) of the membranes resulted in reductions in the concentrations of SDS TTHMs and SDS HAAs. Using a membrane with a 5 kD MWCO, the minimum gaseous ozone concentration required to bring about effective NOM degradation and meet regulatory requirements for chlorinated DBPs was 2.5 g/m(3).

Background and Aim: Oxidative injury occurs as a direct result of hepatitis C virus (HCV) core protein expression both in vitro and in vivo, and may be due to a direct effect on mitochondria. The ketoisocaproic acid (KICA) breath test is a simple, reliable, and noninvasive test to evaluate hepatic mitochondrial function. Albumin dialysis (MARS) is an effective bridge treatment for patients with acute failure superimposed on chronic liver disease. The aim of our study was to evaluate the improvement of mitochondrial function measured by KICA in patients undergoing MARS for acute-on-chronic HCV liver failure., Materials and Methods: Five patients with HCV chronic infection undergoing MARS treatment for acute decompensation were enrolled. Before and after each MARS treatment, patients underwent blood testing for the main hematochemical parameters as well as for mitochondrial function by the KICA breath test and the arterial ketone bodies ratio (AKBR)., Results: MARS treatment effectively decreased the serum level of total bilirubin, bile acids, urea, and ammonium. Moreover, MARS treatment produced an increase in AKBR and in the cumulative percentage of (13)CO(2) recovered in exhaled air 2 hours after KICA ingestion., Conclusion: Liver mitochondrial function appears to be beneficially affected by MARS treatment.

Reactions of O3 with pure and mixed oleic acid particles and bulk solutions were investigated using a thermal desorption particle beam mass spectrometer. The results provide information on the effect of particle matrix on reaction products, mechanisms, and kinetics. The major aerosol products are alpha-acyloxyalkyl hydroperoxides, secondary ozonides, alpha-alkoxyalkyl hydroperoxides, and oxocarboxylic acids formed primarily through reactions of Criegee intermediates with products or with particle matrix compounds. For example, it is estimated that for the reaction of pure oleic acid particles with O3 the aerosol products consist of approximately 68% organic peroxides, 28% 9-oxononanoic acid, and 4% azelaic acid. Although the reaction rate of pure oleic acid particles corresponds to an atmospheric lifetime of minutes, reactions in liquid/solid particle matrices can be orders of magnitude slower. The peroxide products are relatively stable when exposed to matrices typical of atmospheric particles, indicating that the lifetimes of these compounds in the atmosphere may be long enough to allow for long-range transport.

HIV-1 integrase (IN) is an attractive and validated target for the development of novel therapeutics against AIDS. Significant efforts have been devoted to the identification of IN inhibitors using various methods. In this context, through virtual screening of the NCI database and structure-based drug design strategies, we identified several pharmacophoric fragments and incorporated them on various aromatic or heteroaromatic rings. In addition, we designed and synthesized a series of 5-aryl(heteroaryl)-isoxazole-3-carboxylic acids as biological isosteric analogues of beta-diketo acid containing inhibitors of HIV-1 IN and their derivatives. Further computational docking studies were performed to investigate the mode of interactions of the most active ligands with the IN active site. Results suggested that some of the tested compounds could be considered as lead compounds and suitable for further optimization.

Ten water soluble organic acids in PM2.5 of atmosphere in Beijing were determined by capillary electrophoresis, including low molecular weight dicarboxylic acids, aldehyde acids, ketoacids and methanesulfonic acid. The quantified detection limits ranged from 110 to 280 microg/L (extract). The precise of peak area was between 1.7% and 6.0%, and the correlation coefficients of standard curve were 0.995-0.999. The average total concentrations of the ten organic acids in summer, fall and winter were 721 ng/m3, 362 ng/m3 and 325 ng/m3, respectively, which were comparable with some foreign big cities. Oxalic acid was the most abundant species. By exploring seasonal variations of the acids, their source characteristics were simply discussed.

Breath tests for "dynamic" liver function evaluation have been proposed several years ago. A variety of carbon-labelled breath tests for the assessment of mitochondrial, microsomal and cytosolic liver function have been described with the aim to increase data on liver disease staging, prognosis, and response to therapy. In the last years a great interest is developed about the use of breath test for liver mitochondrial function evaluation since it results impaired in a wide range of liver diseases either of genetic or acquired origin. In these cases mitochondrial oxidative metabolism of some substrates, as far as recovery of the hepatic energy state after a metabolic insult, results impaired because of the disfunction of the electron transport chain and/or ATP synthesis. Ketoisocaproic acid and methionine are the best studied carbon-labelled substrates for the investigation of mitochondrial functional damages related to structural alterations that occur in many liver diseases. Although these tests are simple, cost-effective and safe, to date there is still not general approval for their usefulness in clinical settings since they should fulfill several requirements to overcome the drawbacks of traditional quantitative tests. On the other hand, this field is relatively young and further studies are needed in order to assess the suitable substrate for the evaluation of the complex mitochondrial metabolism both in healthy subjects and in patients with liver disease.

The bacteriocin, lacticin 3147, increased isoleucine transamination by Lactococcus lactis IFPL359 in a cheese model system. The formation of alpha-keto-beta-methyl-n-valeric acid and 2-hydroxy-3-methyl-valeric acid increased by three times in cheese slurries at 12 degrees C and cheese aroma intensity increased as well, which corresponded with a higher 2-methylbutanal formation.

Only 2% of the known natural products with acetylenic bonds are alpha-alkynoates. Their polarized, conjugated triple bond is an optimal target for an enzymic hydration. Therefore they are good substrates for the enzymes involved in metabolism of acetylenic compounds, resulting in products that are suitable for bacterial growth. We isolated a Pseudomonas putida strain growing on 2-butynedioate as well as on propynoate, and determined the metabolic pathways of these two alpha-alkynoates. The triple bonds in both compounds were initially hydrated and 2-ketobutandioate as well as 3-ketopropanoate were formed. These two beta-keto acids were decarboxylated resulting in pyruvate and acetaldehyde, respectively. Pyruvate was further hydrolysed mainly to acetate and formate, whereas minor amounts were reduced to lactate. In the other biotransformation, acetaldehyde was oxidized to acetate accompanied by the reduction of 3-ketopropanoate to 3-hydroxypropanoate. Analyses of these metabolic processes were performed by in situ 1H-NMR spectroscopy in 1H2O, although the substrates, propynoate and 2-butynedioate, carried only one or even no detectable protons, respectively. However, while protons from the solvent are incorporated in the course of the pathway, the metabolites can be detected and identified. Therefore a detailed determination of the metabolic process is possible.

Dysfunction in mitochondrial processes has been related to several pathologies. In these disorders, the cell suffers oxidative imbalance that is mostly due to defects in pyruvate metabolism, mitochondrial fatty acids oxidation, the citric acid cycle or electron transport by the mitochondrial respiratory chain. These metabolic alterations produce mitochondrial diseases that have been related to inherited syndromes, such as MERRF or MELAS. The main affected organs are brain, skeletal muscle, kidney, heart and liver, because of the high energetic demand and the oxidative metabolism. Moreover, the relationship between mitochondrial dysfunction and neurodegenerative processes, such as Parkinson disease or Alzheimer disease, as well as ageing, has been shown. Because mitochondrias are the target of several xenobiotics, such as aspirin, AZT or alcohol consumption, mitochondrial impairment has also been proposed as a mechanism of toxicity. Most laboratory tests that are available in the diagnosis of mitochondrial illness are assayed in tissue biopsies and are usually difficult to interpret. Recently, it has been shown that non-invasive techniques, such as nuclear magnetic resonance or the 2-keto[1-(13)C]isocaproic acid breath test, may be useful to assess mitochondrial function. This article attempts to show the laboratory approach to mitochondrial diseases, reviewing new techniques that could be of great value in the research of mitochondrial function, such as the 2-keto[1-(13)C]isocaproic breath test.

A wind tunnel bioassay and video to observe mosquitoes landing on heated glass cylinders were used to test sweat and some derivatives for responses of Anopheles gambiae Giles (Diptera: Culicidae), a highly anthropophilic African species of malaria vector. Filter papers impregnated with human sweat and a diethyl ether extract from the filter papers elicited significantly more landings than a water control (P<0.001). The concentration of lactic acid in the extract was determined by GLC assay, but bioassays of an equivalent dose of lactic acid (from a commercial supplier) did not elicit landings. Chemical analysis of the extract by combined GLC/mass spectrometry indicated the presence of 73 compounds, of which 40 were tentatively identified. The major components of the extract were aliphatic carboxylic acids. An artificial blend of 22 carboxylic acids did not elicit landings. Bioassays of 2-oxopentanoic acid elicited significantly more, landings (P<0.001). The possible importance of oxo-carboxylic acids for host-seeking by anthropophilic mosquitoes is discussed and their use for trapping is suggested.

We report a validated method for the determination of extra- and intracellular [1-(13)C]-alpha-ketoisovalerate ([1-(13)C]-KIV) enrichments by gas chromatography-mass spectrometry. Standardization curves were prepared by enzymatic oxidation of [1-(13)C]-valine enriched standards of known composition. Slopes of [1-(13)C]-valine standardization curves (mean+/-SD: 0.99+/-0.02, n=5) and [1-(13)C]-KIV standardization curves (mean+/-SD: 0.98+/-0.01, n=7) were not significantly different. The method was applied for the determination of [1-(13)C]-KIV enrichments in plasma and tissues during [1-(13)C]-valine infusion in a piglet. [1-(13)C]-KIV enrichment could be determined+/-0.1 MPE (C.V. 1%), and extracellular [1-(13)C]-KIV enrichment was a reliable estimate of intracellular (skeletal muscle, bone growth plate) [1-(13)C]-KIV enrichment.

The development of new or improved analytical methods requires new enzymes. Screening techniques utilizing enrichment cultures and rapid assay methods supported by automated or miniaturized methods are useful tools to detect new enzyme producers. Notably, oxidoreductases are well suited for analytical purposes. The NAD(P)- and oxygen-independent quinoprotein dehydrogenases with a covalently bound redox cofactor can be used advantageously for the development of biosensors. Examples are given of selective enrichment methods used in screening for useful enzyme-producing microorganisms. Enrichment under chemostatic conditions proved to be successful because enzymes with a remarkably high affinity against the analyte could be obtained. This is demonstrated by the screening of a trimethylamine-converting enzyme. The frequently observed high selectivity of these enzymes against the substrate is demonstrated in a few examples. In exploitation of these new oxidoreductases, new analytical methods were developed which are useful for the detection and during monitoring of phenylketonuria (PKU) or maple syrup urine disease (MSUD).

The aim of this work was to study the effects of low energy parenteral diets with different lipid/glucose ratios on rat liver and jejunal mucosa protein synthesis. The studied diets were: L0 (100% glucose, control diet), L25 (25% lipids: 75% glucose), L50 (50% lipids: 50% glucose) and L75 (75% lipids: 25 % glucose). All diets were isoenergetic and isonitrogenated, with a standard amino acid content. The diets were assayed in 93 rats with open femoral fracture immobilized by Kirschner pin insertion. The diets were administered for 4 days. On the fifth day, liver and jejunal mucosa protein synthesis were determined. Highest liver protein synthesis rates were obtained with the diet compositions: lipid/carbohydrate ratio: 25% lipids and 75% carbohydrates (expressed as energy ratio). A higher proportion of lipids significantly decreases liver protein synthesis (p <0.05). Jejunal mucosa protein synthesis followed the same pattern, with the same statistical differences.

The increase in bone mass, muscle bulk, and linear growth that occur during puberty are mediated, at least in part, through the action of sex steroids. By using infusions of nonradioactive tracers of leucine, we have recently shown a significant protein anabolic effect of testosterone in prepubertal boys. The present study was designed to determine whether estrogens can cause similar changes in protein metabolism in females. Seven prepubertal girls (Turner's syndrome, n = 6; hypogonadotropism, n = 1; mean age 12.2 +/- 0.3 yr) were studied. A 4-h infusion of L(-)[1-(13C)]leucine was given, and the isotopic enrichment of [13C]ketoisocaproic acid and 13CO2 were measured in plasma and in breath with use of gas chromatography/mass spectroscopy and an isotope ratio mass spectrometer, respectively. The reciprocal pool model was used for analysis of leucine kinetics. Subjects were then started on ethinyl estradiol orally (n = 5) or depot estradiol parenterally (n = 2). An identical study was repeated 4 weeks later. There were comparable changes in leucine kinetics in all girls studied, hence their data were grouped for analysis. After administration of ethinyl estradiol, there were insignificant changes in the rate of appearance of leucine, an index of proteolysis (+6% +/- 8%); leucine oxidation (-9% +/- 11%); and nonoxidative leucine disposal, an index of whole body protein synthesis (+10% +/- 8%). This is in sharp contrast to the changes found in boys studied similarly after treatment with testosterone (rate of appearance: 17 +/- 6%, P = .036; leucine oxidation -49 +/- 5%, P = 0.004; and nonoxidative leucine disposal +35 +/- 8%, P = 0.009). The lack of anabolic effect on whole-body protein in the girls reported here was observed despite significant increases in plasma insulin-like growth factor I concentrations during 4 h of sampling when comparing the 2 study days, similar to that of the prepubertal boys who were treated with testosterone. In summary, we observed that contrary to androgens, estrogens do not alter estimates of whole-body protein turnover and anabolism in prepubertal humans, despite significant increases in circulating insulin-like growth factor I concentrations. In conclusion, the impact of testosterone on protein metabolism seems to be a direct effect of androgens, independent of aromatization. These findings correlate with the significant differences in muscle bulk between the sexes as children go through puberty.

A simple, rapid, and sensitive spectrophotometric method has been developed for the determination of alpha-keto acids, alpha-diketones, and alpha-ketoaldehydes using o-phenylenediamine as a derivatizing reagent in acidic media to yield chromophoric quinoxaline derivatives. Application to the determination of alpha-ketobutyrate and the rate of its formation from isomerization of vinylglycolate catalyzed by mandelate racemase is described. Under optimal conditions 1 to 4 mM o-phenylenediamine in 80% acetic acid was incubated at 50 degrees C for 10 min with the alpha-dicarbonyl compounds. Solutions of alpha-ketobutyrate as low as 5 microM in the buffer solution can be measured quantitatively. This method has also been used for determining the rate of formation of the phenylglyoxalate from (S)-mandelate catalyzed by (S)-mandelate dehydrogenase.

Two experiments with weanling pigs were conducted to study the effects on growth and immune responses of excess dietary L-leucine (LEU) and dietary supplementation with the LEU catabolites, alpha-ketoisocaproic acid (KIC) and beta-hydroxymethyl butyrate (HMB). In Exp. 1, 80 pigs were randomly allocated according to initial BW and ancestry to five replications of four dietary treatments (four pigs/pen). The control diet contained wheat, oat groats, menhaden fish meal, and dried whey and provided 1.12% LEU. Treatment diets were the control plus 1.12% LEU, 1.12% KIC, or .4% HMB. The experiment lasted 6 wk. In Exp. 2, 36 pigs were randomly allocated to nine replications of four dietary treatments in a 2 x 2 factorial arrangement. Treatments consisted of two concentrations of dietary LEU and a daily i.m. injection of dexamethasone (DEX) or saline. Pigs were fed a control corn-soybean meal and dried whey diet (1.56% LEU) or the control diet plus 1.56% of crystalline LEU. Pigs were individually penned and the experiment lasted 4 wk. Growth performance, plasma free amino acids, plasma urea nitrogen, and humoral and cellular immune responses were measured. Results indicated that LEU concentrations in practical diets and supplementation with KIC and HMB (Exp. 1) did not detrimentally affect growth and immune response. The high LEU concentration and DEX injection used in Exp. 2, however, were detrimental to both growth and immune response.

A fast and sensitive method for the single-run quantification of various 2-oxo acids including 2-ketoglutarate, glyoxylate and pyruvate is described. It ensures good separation of peaks with minor interference by other substances. The 2-oxo acid derivatives are measured photometrically at 324 nm after derivatization with phenylhydrazine and subsequent isocratic separation with ethanolic phosphate buffer on a C18 reversed-phase HPLC column. Recovery was found to be complete [range: 96.3 +/- 5.6% (pyruvate) to 104.8 +/- 5.2% (2-ketoglutarate)]. Detection limits ranged from less than 0.1 mumol/l (glyoxylate) to 0.25 mumol/l (2-ketoglutarate and pyruvate). Results for all substances examined showed good linearity with correlations (r2) of equal to or better than 0.998.

The primary objective of the present study was to assess whether the inhibitory effect of leucine in rat hearts on protein degradation is mediated by leucine itself, alpha-ketoisocaproate (alpha-KIC) or the decarboxylated products of leucine. Protein degradation, as measured by the release of phenylalanine, was inhibited by 1 mM leucine in hearts supplied with glucose, despite low intracellular alpha-KIC concentration (6.3 +/- 0.5 microM). The inhibition of protein degradation by leucine occurred in hearts supplied with pyruvate, a substrate that completely abolished leucine decarboxylation. Under this condition, leucine was transaminated to alpha-KIC. Since the transamination could not be inhibited more than 38% by 10 mM L-cycloserine, it was difficult to exclude that leucine exerted its inhibitory effect via transamination to alpha-KIC. A clear correlation between protein degradation and intracellular leucine or alpha-KIC concentration could not be established. However, a high concentration of leucine (1 mM) or alpha-KIC (0.3 mM) in the perfusate inhibited protein degradation by 30 and 20%, respectively. This suggested that both leucine and alpha-KIC act on the plasma membrane regulatory sites. Of the various structural analogues of leucine that were tested, only L-leucinol inhibited protein degradation. This inhibition could be attributed to a direct lysosomotropic effect of this amino alcohol. The results indicate that high concentrations of extracellular leucine and alpha-KIC appear to inhibit heart protein degradation in vitro.

In order to assess the combined and separate effects of pancreas and kidney transplant on whole-body protein metabolism, 9 insulin-dependent diabetic-uremic patients (IDDUP), 14 patients after combined kidney-pancreas transplantation (KP-Tx), and 6 insulin-dependent diabetic patients with isolated kidney transplant (K-Tx), were studied in the basal postabsorptive state and during euglycemic hyperinsulinemia (study 1). [1-14C]Leucine infusion and indirect calorimetry were utilized to assess leucine metabolism. The subjects were studied again with a combined infusion of insulin and amino acids, given to mimic postprandial amino acid levels (study 2). In the basal state, IDDUP demonstrated with respect to normal subjects (CON): (a) higher free-insulin concentration (17.8 +/- 2.8 vs. 6.8 +/- 1.1 microU/ml, P < 0.01) (107 +/- 17 vs. 41 +/- 7 pM); (b) reduced plasma leucine (92 +/- 9 vs. 124 +/- 2 microM, P < 0.05), branched chain amino acids (BCAA) (297 +/- 34 vs. 416 +/- 10 microM, P < 0.05), endogenous leucine flux (ELF) (28.7 +/- 0.8 vs. 39.5 +/- 0.7 mumol.m-2.min-1, P < 0.01) and nonoxidative leucine disposal (NOLD) (20.7 +/- 0.2 vs. 32.0 +/- 0.7 mumol.m-2. min-1, P < 0.01); (c) similar leucine oxidation (LO) (8.0 +/- 0.1 vs. 7.5 +/- 0.1 mumol.m-2.min-1; P = NS). Both KP-Tx and K-Tx patients showed a complete normalization of plasma leucine (116 +/- 5 and 107 +/- 9 microM), ELF (38.1 +/- 0.1 and 38.5 +/- 0.9 mumol.m-2.min-1), and NOLD (28.3 +/- 0.6 and 31.0 +/- 1.3 mumol.m-2.min-1) (P = NS vs, CON). During hyperinsulinemia (study 1), IDDUP showed a defective decrease of leucine (42% vs. 53%; P < 0.05), BCAA (38% vs. 47%, P < 0.05), ELF (28% vs. 33%, P < 0.05), and LO (0% vs. 32%, P < 0.05) with respect to CON. Isolated kidney transplant reverted the defective inhibition of ELF (34%, P = NS vs. CON) of IDDUP, but not the inhibition of LO (18%, P < 0.05 vs. CON) by insulin. Combined kidney and pancreas transplanation normalized all kinetic parameters of insulin-mediated protein turnover. During combined hyperinsulinemia and hyperaminoacidemia (study 2), IDDUP showed a defective stimulation of NOLD (27.9 +/- 0.7 vs. 36.1 +/- 0.8 mumol.m-2.min-1, P < 0.01 compared to CON), which was normalized by transplantation (44.3 +/- 0.8 mumol.m-2.min-1).

An extremely sensitive fluorimetric HPLC method used to determine in vivo cerebral alpha-ketoisocaproic acid (KIC) concentration with less than 2 g of tissue (e.g., a single rat brain, approximately 1.8 g) is reported. Removal of unwanted lipids and amino acids and isolation of alpha-ketoacids in an optimal derivatization buffer are achieved by C18 solid-phase extraction of the acid-soluble fraction of brain tissue. Quantitation of KIC to the femtomole level is accomplished by reversed-phase HPLC using 4,5-dimethoxy-1,2-diaminobenzene precolumn fluorescence derivatization and on-line fluorescence detection. These techniques are applicable to femtomole quantitation of other alpha-ketoacids in various tissue and blood matrices. Combination of this fluorimetric method with simple nonchromatographic procedures to measure in vivo cerebral [1-14C]alpha-ketoisocaproic acid radioactivities in tissue provides estimates of specific activities.

According to previous reports, branched chain ketoacids (BCKAs) are undetectable in liver and brain. A procedure is described for HPLC analysis of tissue concentrations of BCKA that achieves 80% recovery of labeled 2-ketoisocaproate (KIC) added to frozen powdered liver. In normal overnight-fasted rats, substantial hepatic uptake was demonstrable in the order KIC > 2-ketoisovalerate (KIV) congruent to 2-keto-3-methylvalerate (KMV). All three BCKAs were very low in freeze-clamped liver. Liver concentrations were only 15% to 22% of the mean of portal vein, hepatic vein, and hepatic artery (aorta) concentrations. In view of the probable contribution to total liver content of extracellular BCKAs, the intracellular concentrations in liver tissue must be vanishingly low. Somewhat higher amounts were present in freeze-clamped kidney and heart tissue, and significant uptake of KIC and KMV by kidney was found. Brain released KIC. Muscle contained concentrations of BCKA that were 35% to 72% of concentrations in plasma, and it released all three BCKAs. Similar results were obtained from tissues in overnight-fasted dogs, except that even lower levels were found in freeze-clamped brain (sampled before circulation ceased) than were found in the liver. Plasma levels in dogs were similar to those of rats. In both species the tissue-to-plasma ratios were higher for KIV than for KMV or KIC. Remarkably low BCKA levels in liver and brain are unexplained, particularly in light of evidence that BCKAs gain access to cells in both organs.

Threonine dehydratase, a key enzyme leading to the biosynthesis of isoleucine, catalyzes the production of 2-ketobutyrate from threonine. An uv/fluorometric HPLC assay for threonine dehydratase has been developed that involves derivatization of the 2-ketoacids produced by the enzyme using a specific derivatizing agent, o-phenylenediamine. The derivatized ketoacids can be detected spectrophotometrically or fluorometrically. This novel assay is over 1000-fold more sensitive than the commonly used dinitrophenyl hydrazine derivatization assay. In addition, the HPLC assay allows the identification of the reaction product(s), and can be used to examine the reaction using mixtures of the two substrates, threonine and serine.

A new procedure for the assay of D-amino acid oxidase activity has been developed. alpha-Ketoisovaleric acid, derived from D-valine, was estimated by high-performance liquid chromatography after reaction with o-phenylenediamine to give the corresponding quinoxalinol derivative. alpha-Ketovaleric acid was used as an internal standard to ensure the reproducibility of the method. As an example of application, kidney cortex homogenates were analyzed for their D-amino acid oxidase activity. The advantages of the presented procedure for the determination of the enzymatic activity in biological samples compared with previously reported procedures are discussed.

Many investigators are now using stable isotopes in place of radioactive isotopes because of ethical considerations in human research. Our laboratory has had a long history in the development of an ovine model for the study of the physiological and biochemical changes during pregnancy. We wanted to extend some of the hypotheses and experimental protocols developed while using this model system to the human. As a first step in this process, we carried out infusions using a mixture of both 14C and 13C isotopes of the essential amino acid L-leucine. Results from this study showed that turn-over rates calculated using the two isotopes were equal within experimental error (8.99 +/- 0.45 and 8.97 +/- 0.52 mumol/kg.min, respectively). A key step in the development of the techniques for this study involved the use of tert.-butyldimethylsilyl derivatives for the amino acids. Because of the strong M-57 peak seen in the mass spectra of these compounds, we were able to use a relatively inexpensive Hewlett-Packard mass-selective detector for these determinations. Enrichments in triplicate measurements of the same sample had a precision of +/- 0.03%. Similar precision data were obtained in enrichment measurements of the keto acids derived from the amino acids. The combination of the speed of the analysis and the excellent precision have provided us with the opportunity to study uptake and loss across an organ system (i.e. placenta, liver, etc). This tool is now being used to study the detailed flow of amino acids across the placenta during both normal and abnormal pregnancies.

Cholecystectomy has been hypothesized to cause increased levels of potentially harmful secondary bile acids due to prolonged exposure of primary bile acids to intestinal bacteria. In the present study, we analyzed duodenal bile of the same patients before and after cholecystectomy for keto bile acids and hydroxy bile acids. The ratios of individual keto bile acids to their corresponding precursor hydroxy bile acids were not significantly different before and after cholecystectomy. Keto bile acids constituted 2.5 +/- 1.3 and 2.2 +/- 0.9 mol% of hydroxy bile acids in duodenal bile. 3 alpha-Hydroxy-12-oxo-5 beta-cholanoic acid derived from deoxycholic acid was the main keto bile acid in bile contributing 80 mol% to total keto bile acids. There was a strong positive correlation between 3 alpha-hydroxy-12-oxo-5 beta-cholanoic acid and its precursor deoxycholic acid in bile (r = 0.88; p less than 0.0005). Our findings prove that 7 weeks after cholecystectomy the percentage of keto bile acids in bile as compared to hydroxy bile acids is not increased.

Profiles of metabolic alpha-keto acids were determined by a high-performance liquid chromatographic method and applied to characterization of oral black-pigmented Bacteroides. Each bacterial strain was incubated with amino acids in a chemically defined medium. After production alpha-keto acids were purified by hydrazide gel column treatment and converted to u.v.-absorbing derivatives. They were analysed by reversed-phase ion-pair chromatography. Black-pigmented Bacteroides species were differentiated into two groups according to production of aromatic alpha-keto acids. Bacteroides gingivalis, B. endodontalis and B. loescheii produced both p-hydroxyphenylpyruvic and phenylpyruvic acids. However, no such alpha-keto acids were produced by B. levii, B. intermedius and B. denticola. In addition, production profiles of several aliphatic alpha-keto acids (alpha-ketoglutaric, pyruvic, alpha-ketobutyric, alpha-ketoisovaleric, alpha-ketoisocaproic, and alpha-keto-beta-methylvaleric acids) separated each individual species in such groups. The present study offers useful chemotaxonomic information on amino acid metabolic activity of oral black-pigmented Bacteroides species.

A simple, highly sensitive method for the simultaneous determination of arrays of carbon-centered radicals in aqueous systems is described. Radicals are efficiently trapped by an amino-nitroxide to form stable products which are then reacted with fluorescamine to produce highly fluorescent adducts. The adducts are easily separated by reversed-phase high performance liquid chromatography. The detection limit for individual radical adducts (0.5 to 2 nM) is two to three orders of magnitude lower than those of current methods employing electron paramagnetic resonance detection. Results on the photolysis of ketones and alpha-keto acids demonstrate the potential of this technique. This approach should be widely applicable to the study of radical processes in biological and chemical systems.

Male rats were fed either a 10% casein diet or a 10% casein diet supplemented with 5% leucine, and the levels of alpha-ketoisocaproic acid and ketone bodies were analyzed in blood and the liver. alpha-Ketoisocaproic acid in plasma increased significantly on the 1st day of feeding the leucine-supplemented diet and was maintained on the high level during the experimental period of 7 days. Other branched chain alpha-keto acids tended to decrease in plasma. beta-Hydroxybutyric acid in blood doubled its amount on the 1st day on the high leucine diet and was maintained high during the experimental period. The sum of acetoacetic acid and beta-hydroxybutyric acid in blood was kept on a level twice as high as that of the control group. Ketone bodies in the liver were similarly increased and the sum of acetoacetic acid and beta-hydroxybutyric acid in the liver corresponded to the level found inhibitory to NAD biosynthesis in in vitro system [YAMADA, O., et al.: Int. J. Vit. Nutr. Res. 53, 184 (1983)].